1. Field of the Invention
The present invention concerns methods and means for the identification and use of modulators of β-amyloid (Aβ) levels obtained by the proteolytic processing of the β-amyloid precursor protein, APP.
2. Description of the Related Art
A number of important neurological diseases, including Alzheimer's disease (AD), cerebral amyloid angiopathy (CAA), and prion-mediated diseases are characterized by the deposition of aggregated proteins, referred to amyloid, in the central nervous system (CNS)(for reviews, see Glenner et al., J. Neurol. Sci. 94:1–28 [1989]; Haan et al., Clin. Neurol. Neurosurg. 92(4):305–310 [1990]). These highly insoluble aggregates are composed of nonbranching, fibrillar proteins with the common characteristic of β-pleated sheet conformation. In the CNS, amyloid can be present in cerebral and meningeal blood vessels (cerebrovascular deposits) and in the brain parenchyma (plaques). Neuropathological studies in human and animal models indicate that cells proximal to amyloid deposits are disturbed in their normal functions (Mandybur, Acta Neuropathol. 78:329–331 [1989]; Kawai et al., Brain Res. 623:142–146 [1993]; Martin et al., Am. J. Pathol. 145:1348–1381 [1994]; Kalaria et al., Neuroreport 6:477–480 [1995]; Masliah et al., J. Neurosci. 16:5795–5811 [1996]; Selkoe, J. Biol. Chem. 271:18295–18298 [1996]; Hardy, Trends Neurosci 20:154–159 [1997]).
AD and CAA share biochemical and neuropathological markers, but differ somewhat in the extent and location of amyloid deposits as well as in the symptoms exhibited by affected individuals. The neurodegenerative process of AD, the most common neurodegenerative disorder worldwide, is characterized by the progressive and irreversible deafferentation of the limbic system, association neocortex, and basal forebrain accompanied by neuritic plaque and tangle formation (for a review, see Terry et al., “Structural alteration in Alzheimer's disease,” In: Alzheimer's disease, Terry et al. Eds., 1994, pp. 179–196, Raven Press, New York). Dystrophic neurites, as well as reactive astocytes and microglia, are associated with these amyloid-associated neuritic plaques. Although the neuritic population in any given plaque is mixed, the plaques generally are composed of spherical neurites that contain synaptic proteins, APP (type I), and fusiform neurites containing cytoskeletal proteins and paired helical filaments (PHF; type II).
CAA patients display various vascular syndromes, of which the most documented is cerebral parenchymal hemorrhage. Cerebral parenchymal hemorrhage is the result of extensive amyloid deposition within cerebral vessels (Hardy, Trends Neurosci 20:154–159 [1997]; Haan et al., Clin. Neurol. Neurosurg. 92:305–310 [1990]; Terry et al., [1994] supra; Vinters, Stroke 18:211–224 [1987]; Itoh et al., J. Neurosurgical Sci. 116:135–141 [1993]; Yamada et al., J. Neurol. Neruosurg. Psychiatry 56:543–547 [1993]; Greenberg et al., Neurology 43:2073–2079 [1993]; Levy et al., Science 248:1124–1126 [1990]). In some familial CAA cases, dementia was noted before the onset of hemorrhages, suggesting the possibility that cerebrovascular amyloid deposits may also interfere with cognitive functions.
Both AD and CAA are characterized by the accumulation of senile plaques in the brains of the affected individuals. The main amyloid components is the amyloid β protein (Aβ), also referred to as amyloid β or β-amyloid peptide, derived from proteolytic processing of the β-amyloid precursor protein, β-APP or simply APP. For review in connection with Aβ see, Selkoe, D. J. Nature 399: A23–A31 (1999). Aβ is produced by proteolytic cleavage of an integral membrane protein, termed the β-amyloid precursor protein (βAPP).
The Aβ peptide, which is generated from APP by two putative secretases, is present at low levels in the normal CNS and blood. Two major variants, Aβ1-40 and Aβ1-42 are produced by alternative carboxy-terminal truncation of APP (Selkoe et al. [1988] Proc. Natl. Acad. Sci. USA 85:7341–7345; Selkoe [1993] Trends Neurosci 16:403–409). Aβ1-42 is the more fibrillogenic and more abundant of the two peptides in amyloid deposits of both AD and CAA. In addition to the amyloid deposits in AD cases described above, most AD cases are also associated with amyloid deposition in the vascular walls (Hardy [1997], supra; Haan et al. [1990], supra; Terry et al., [1994] supra; Vinters [1987], supra; Itoh, et al. [1993], supra; Yamada et al. [1993], supra; Greenberg et al. [1993], supra; Levy et al. [1990], supra). These vascular lesions are the hallmark of CAA, which can exist in the absence of AD.
The precise mechanisms by which neuritic plaques are formed and the relationship of plaque formation to the AD-associated, and CAA-associated neurodegenerative processes are not well defined. However, evidence indicates that dysregulated expression and/or processing of APP gene products or derivatives of these gene products are involved in the pathophysiological process leading to neurodegeneration and plaque formation. For example, missense mutations in APP are tightly linked to autosomal dominant forms of AD (Hardy [1994] Clin. Geriatr. Med. 10:239–247; Mann et al. [1992] Neurodegeneration 1:201–215). The role of APP in neurodegenerative diseases is further implicated by the observation that persons with Down's syndrome who carry an additional copy of the human APP (HAPP) gene on their third chromosome 21 show an overexpression of hAPP (Goodison et al. [1993] J. Neuropathol. Exp. Neurol. 52:192–198; Oyama, et al. [1994] J. Neurochem. 62:1062–1066) as well as a prominent tendency to develop AD-type pathology early in life (Wisniewski et al. [1985] Ann. Neurol. 17:278–282). Mutations in Aβ are linked to CAA associated with hereditary cerebral hemorrhage with amyloidosis (Dutch HCHWA)(Levy, et al. [1990], supra), in which amyloid deposits preferentially occur in the cerebrovascular wall with some occurrence of diffuse plaques (Maat-Schieman et al. [1994] Acta Neuropathol. 88:371–8; Wartendorff et al. [1995] J. Neurol. Neurosurg. Psychiatry 58:699–705). A number of hAPP point mutations that are tightly associated with the development of familial AD encode amino acid changes close to either side of the Aβ peptide (for a review, see, e.g., Lannfelt et al. [1994] Biochem. Soc. Trans. 22:176–179; Clark et al. [1993] Arch. Neurol. 50:1164–1172). Finally, in vitro studies indicate that aggregated Aβ can induce neurodegeneration (see, e.g., Pike et al. (1995) J. Neurochem. 64:253–265).
APP is a glycosylated, single-membrane-spanning protein expressed in a wide variety of cells in many mammalian tissues. Examples of specific isotypes of APP which are currently known to exist in humans are the 695-amino acid polypeptide (APP695) described by Kang et al. (1987) Nature 325:733–736, which is designated as the “normal” APP. A 751-amino acid polypeptide (APP751) has been described by Ponte et al. (1988) Nature 331:525–527 and Tanzi et al. (1988) Nature 331:528–530. A 770-amino acid isotype of APP (APP770) is described in Kitaguchi et al. (1988) Nature 331:530–532. A number of specific variants of APP have also been described having mutations which can differ in both position and phenotype. A general review of such mutations is pivoted in Hardy (1992) Nature Genet. 1:233–235. A mutation of particular interest is designated the “Swedish” mutation where the normal Lys-Met residues at positions 595 and 596 are replaced by Asn-Leu. This mutation is located directly upstream of the normal β-secretase cleavage site of APP, which occurs between residues 596 and 597 of the 695 isotype.
APP is post-translationally processed by several proteolytic pathways resulting in the secretion of various fragments or intracellular fragmentation and degradation. F. Checler, J. Neurochem. 65:1431–1444 (1995). The combined activity of β-secretase and γ-secretase on APP releases an intact β-amyloid peptide (Aβ), which is a major constituent of amyloid plaques. Initial cleavage of APP by β-secretase generates soluble APPβ and membrane-associated β-CTF that can be further processed by γ-secretase to generate a 40 or a 42 amino acid peptide (Aβ40 or Aβ42). Alternatively, APP processing by α-secretase leads to the formation of soluble APPα and membrane associated α-CTF the latter being a substrate for γ-secretase to generate the non-amyloidogenic p3. Aβ is an approximately 43 amino acid peptide, which comprises residues 597–640 of the 695 amino acid isotype of APP. Internal cleavage of Aβ by a α-secretase inhibits the release of the full-length Aβ peptide. Although the extent of pathogenic involvement of the secretases in AD progression is not fully elucidated, these proteolytic events are known to either promote or inhibit Aβ formation, and thus are thought to be good therapeutic candidates for AD.
The polytopic transmembrane protein presenilin has been strongly implicated in γ-secretase activity (for review see Haass and De Strooper, Science 286: 916–919 [1999]). Mutagenesis of two transmembrane aspartates of presenilin led to the inactivation of γ-secretase activity in cellular assays (Wolfe et al., Nature 398: 513–517 [1999]). As a result, both α- and β-CTFs accumulated and Aβ formation was significantly decreased. Similar effects were seen upon inhibition of γ-secretase using substrate analogs (Wolfe et al., J. Med. Chem. 41: 6–9 [1998]). While it remains to be determined whether presenilin is sufficient as γ-secretase or whether it requires another unique co-factor of so far unknown nature to exert its function Presenilin 1 and γ-secretase activity have recently been shown to co-precipitate from membrane extracts (Li et al. Proc. Natl. Acad. Sci. USA 97(11):6138–43 [2000]).
As discussed above, there are at least two proteases involved in the generation of Aβ, referred to as β- and γ-secretases (Citron et al., Neuron 17:171–179 [1996]; Seubert et al., Nature 361:260–263 [1993]; Cai et al., Science 259:514–516 [1993]; and Citron et al., Neuron 14:661–670 [1995]). There have been intense efforts in recent years to identify and characterize these enzymes. Recently five independent groups have reported cloning and characterization of genes corresponding to a β-secretase (Vassar et al., Science 286: 735–741 [1999]; Yan et al., Nature 402: 533–537 [1999]; Sinha et al., Nature 402: 537–540 [1999]; Hussain et al., Mol. Cell. Neurosci. 14: 419–427 [1999]; Lin et al. Proc. Natl. Acad. Sci. USA 97: 1456–1460 [2000]). The membrane-bound aspartyl protease has been variously referred to as β-site APP-cleaving enzyme (BACE), Aspartyl protease-2 (Asp2), memapsin 2 or simply as β-secretase. However, the deduced amino acid sequence of the polypeptide chain reported by all five groups is identical. The cloned enzyme possesses many of the characteristics expected of an authentic β-secretase. In particular, BACE overexpression resulted in an increase in both β-NTF and Aβ levels while suppression of BACE with antisense oligonucleotides led to a significant reduction of these cleavage products. As predicted for the genuine β-secretase, the Swedish double mutant of APP (APPsw, Mullan et al., Nature Genetics 1: 345–347 [1992]; Citron et al, Nature 360: 672–674 [1992]; Cai et al., Science 259: 514–516 [1993]) was cleaved more efficiently by BACE. Taken together, these results have led to the notion that BACE is the main β-secretase activity.
A close homolog of BACE, designated DRAP or BACE2, has been described recently (Acquati et al., FEBS Lett. 468: 59–64 [2000], GenBank accession numbers for the human and mouse cDNA sequences: AF050171 and AF051150, respectively; Bennett et al., J. Biol. Chem. 275:37712–7 [2000]). BACE and BACE2 share 64% amino acid similarity but the role of BACE2 in APP processing has not yet been elucidated. Strikingly, BACE2 expression in brain appears to be very low and this observation has contributed to the assumption that BACE2's role in β-secretase cleavage might only be minor (Bennett et al., ibid).